1. Field of the Invention
The invention relates to bacterial antibiotic resistance. More particularly, the invention relates to compositions and methods for overcoming bacterial antibiotic resistance.
2. Background of the Invention
Bacterial antibiotic resistance has become one of the most important threats to modern health care. Cohen, Science 257:1051-1055 (1992) discloses that infections caused by resistant bacteria frequently result in longer hospital stays, higher mortality and increased cost of treatment. Neu, Science 257:1064-1073 (1992) discloses that the need for new antibiotics will continue to escalate because bacteria have a remarkable ability to develop resistance to new agents rendering them quickly ineffective.
The present crisis has prompted various efforts to elucidate the mechanisms responsible for bacterial resistance. Coulton et al. Progress in Medicinal Chemistry 31:297-349 (1994) teach that the widespread use of penicillins and cephalosporins has resulted in the emergence of xcex2-lactamases, a family of bacterial enzymes that catalyze the hydrolysis of the xcex2-lactam ring common to presently used antibiotics. More recently, Dudley, Pharmacotherapy 15: 9S14S (1995) has disclosed that resistance mediated by xcex2-lactamases is a critical aspect at the core of the development of bacterial antibiotic resistance.
Attempts to address this problem through the development of xcex2-lactamase inhibitors have had limited success. Sutherland, Trends Pharmacol Sci 12: 227-232 (1991) discusses the development of the first clinically useful xcex2-lactamase inhibitor, clavulanic acid, which is a metabolite of Streptomyces clavuligerus. Coulton et al. (supra) disclose two other such semi-synthetic inhibitors, sulbactam and tazobactam presently available. Coulton et al. (supra) also teach that in combination with xcex2-lactamase-susceptible antibiotics, xcex2-lactamase inhibitors prevent antibiotic inactivation by xcex2-lactamase enzymes, thereby producing a synergistic effect against xcex2-lactamase producing bacteria.
Rahil and Pratt, Biochem. J. 275: 793-795 (1991), and Li et al., Bioorg. Med. Chem. 5: 1783-1788 (1997) teach that xcex2-lactamase enzymes are inhibited by phosphonate monoesters. Song and Kluger, Bioorg. Med. Chem. Lett., 4, 1225-1228 (1994), teaches that E. Coli RTEM xcex2-lactamase is inhibited by benzylpenicillin methyl phosphate.
Laird and Spence, J. C. S. Perkin Trans. II 1434 (1973), Kazlauskas and Whitesides, J. Org. Chem. 50: 1069-1076 (1985), Chantrenne, Compte. Rend. Trav. Lab. Carlsberg Ser. Chim. 26: 297 (1948), and Maracek and Griffith, J. Am. Chem. Soc. 92: 917-921 (1970), report synthesis and solvolysis studies of acyl phosphate and acyl phosphonate compounds. Kluger et al., Can. J. Chem. 74: 2395-2400 discloses that aminoacyl phosphates are useful as biomimetically activated amino acids.
The availability of only a few compounds however, is insufficient to counter the constantly increasing diversity of xcex2-lactamases for which a variety of novel and distinct inhibitors has become a necessity. There is, therefore, a need for the ability to identify new xcex2-lactamase inhibitors. The development of fully synthetic inhibitors would greatly facilitate meeting this need. Ideally, certain embodiments of such inhibitors would also bind bacterial DD-peptidases, thus potentially acting both as xcex2-lactamase inhibitors and as antibiotic agents.
The invention provides novel xcex2-lactamase inhibitors, which are structurally unrelated to the natural product and semi-synthetic xcex2-lactamase inhibitors presently available, and which do not possess xcex2-lactam pharmacophore. These new inhibitors are preferably fully synthetic, allowing ready access to a wide variety of structurally related analogs. Certain embodiments of these new inhibitors also bind bacterial DD-peptidases, and thus potentially act both as xcex2-lactamase inhibitors and as antibiotics.
In a first aspect, the invention provides novel acyl phosphate and acyl phosphonate xcex2-lactamase inhibitors. Preferably, such inhibitors have the general mixed anhydride structure of Formula I: 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided, however, that when Y is Z, then Z is not phosphonyl; and further provided that when Y is OZ and Z is phenyl, then X is not methyl or phenyl; when Y is OZ and Z is alkyl or adenosyl, then X is not xcex1-aminoalkyl; and when Y is OZ and Z is benzoyl, then X is not phenyl.
In certain preferred embodiments, the mixed anhydride is an acyl phosphate, and Y is thus OZ. In certain other preferred embodiments, the mixed anhydride is an acyl phosphonate, and Y is thus Z.
In certain preferred embodiments, Y and X are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted; provided that X is not phenylethene.
In a second aspect, the invention provides pharmaceutical compositions comprising an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor and a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, such inhibitors have the general mixed anhydride structure (I): 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
In certain preferred embodiments, Y and X are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted.
In a third aspect, the invention provides methods for inhibiting in vitro or in vivo xcex2-lactamase activity, such methods comprising administering an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor. Preferably, such inhibitors have the general mixed anhydride structure of Formula I: 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
In certain preferred embodiments, X and Y are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted
In a fourth aspect, the invention provides a method for inhibiting bacterial growth, the method comprising administering an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor. Preferably, such inhibitors have the general mixed anhydride structure of Formula I: 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
In certain preferred embodiments, X and Y are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted
In one embodiment of such a method, xcex2-lactamase inhibitor according to the invention is co-administered with an antibiotic. In another embodiment the xcex2-lactamase inhibitor according to the invention has antibiotic activity, and can thus either be administered alone or be co-administered with another antibiotic agent.
The invention relates to bacterial antibiotic resistance. More particularly, the invention relates to compositions and methods for overcoming bacterial antibiotic resistance.
The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.
The invention provides novel xcex2-lactamase inhibitors, which are structurally unrelated to the natural product and semi-synthetic xcex2-lactamase inhibitors presently available, and which do not possess a xcex2-lactam pharmacophore. These new inhibitors are preferably fully synthetic, allowing ready access to a wide variety of structurally related analogs. Certain embodiments of these new inhibitors also bind bacterial DD-peptidases, and thus potentially act both as xcex2-lactamase inhibitors and as antibiotics.
For purposes of the present invention, the following definitions will be used:
As used herein, the term xe2x80x9cxcex2-lactamasexe2x80x9d denotes a protein capable of inactivation of a xcex2-lactam antibiotic. In one preferred embodiment, the xcex2-lactamase is an enzyme which catalyzes the hydrolysis of the xcex2-lactam ring of a xcex2-lactam antibiotic. In certain preferred embodiments, the xcex2-lactamase is microbial. In certain preferred embodiments, the xcex2-lactamase is a serine xcex2-lactamase. In certain other preferred embodiments, the xcex2-lactamase is a zinc xcex2-lactamase. The terms xe2x80x9cclass Axe2x80x9d, xe2x80x9cclass Bxe2x80x9d, xe2x80x9cclass Cxe2x80x9d, and xe2x80x9cclass Dxe2x80x9d xcex2-lactamases are understood by those skilled in the art and can be found described in Waley, The Chemistry of xcex2-Lactamase, Page Ed., Chapman and Hall, London, (1992) 198-228. In particularly preferred embodiments, the xcex2-lactamase is class C xcex2-lactamase of Enterobacter cloacae P99 (hereinafter P99 xcex2-lactamase), or class A xcex2-lactamase of the TEM-2 plasmid (hereinafter TEM xcex2-lactamase).
As used herein, the term xe2x80x9cxcex2-lactamase inhibitorxe2x80x9d is used to identify a compound having a structure as defined herein, which is capable of inhibiting xcex2-lactamase activity. Inhibiting xcex2-lactamase activity means inhibiting the activity of a class A, B, C, or class D xcex2-lactamase. Preferably, such inhibition should be at a 50% inhibition concentration below 100 micrograms/mL, more preferably below 30 micrograms/mL and most preferably below 10 micrograms/mL.
In some embodiments of the invention, the xcex2-lactamase inhibitor is also capable of acting as an antibiotic, for example, by inhibiting bacterial cell-wall cross-linking enzymes. Thus, the term xcex2-lactamase inhibitor is intended to encompass such dual-acting inhibitors. In certain preferred embodiments, the xcex2-lactamase inhibitor is capable of inhibiting D-alanyl-D-alanine-carboxy-peptidases/transpeptidases (hereinafter DD-peptidases). The term xe2x80x9cDD-peptidasexe2x80x9d is used in its usual sense to denote penicillin-binding proteins involved in bacterial cell wall biosynthesis (e.g., Ghysen, Prospect. Biotechnol. 128:67-95 (1987)). In certain particularly preferred embodiments, the D-alanyl-D-alanine-carboxy-peptidases/transpeptidase inhibited is the Streptomyces R61 DD-peptidase.
The term xe2x80x9calkylxe2x80x9d as employed herein refers to straight and branched chain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8 carbon atoms, which may be optionally substituted with one, two or three substituents. Unless otherwise specified, the alkyl group may be saturated, unsaturated, or partially unsaturated. As used herein, therefore, the term xe2x80x9calkylxe2x80x9d is specifically intended to include alkenyl and alkynyl groups, as well as saturated alkyl groups. Preferred alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, hexyl, vinyl, allyl, isobutenyl, ethynyl, and propynyl.
The term xe2x80x9calkylenexe2x80x9d as employed herein refers to saturated, unsaturated, and partially unsaturated groups having from 1 to 8 carbon atoms, preferably 1-6 carbon atoms, more preferably 1-4 carbon atoms, and most preferably 1-2 carbon atoms, positioned between and connecting two other substituents. Preferred alkylene groups include, without limitation, methylene, ethylene, and ethene. For purposes of the invention, an alkylene group preferably refers to a portion of a cyclic structure.
As employed herein, a xe2x80x9csubstitutedxe2x80x9d alkyl, cycloalkyl, aryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, amino, alkylcarboxamido, arylcarboxamido, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, cyano, and alkylaminocarbonyl groups.
The term xe2x80x9ccycloalkylxe2x80x9d as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12, preferably 3 to 8, more preferably 3 to 6 carbons, wherein one or two ring positions may be substituted with an oxo group, and wherein the cycloalkyl group additionally may be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexanone, cycloheptyl, and cyclooctyl. A xe2x80x9ccycloalkylenexe2x80x9d group is a cycloalkyl group positioned between and connecting two other substituents. Preferred cycloalkylene groups include, without limitation, cyclohexylene, cyclopentylene, and cyclobutylene.
An xe2x80x9carylxe2x80x9d group is a C6-C14 aromatic moiety comprising one to three aromatic rings, which may be optionally substituted. Preferably, the aryl group is a C6-C10 aryl group. Preferred aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An xe2x80x9caralkylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d group comprises an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted. Preferably, the aralkyl group is (C6-10)ar(C1-6)alkyl, including, without limitation, benzyl, phenethyl, and naphthylmethyl. An xe2x80x9calkarylxe2x80x9d or xe2x80x9calkylarylxe2x80x9d group is an aryl group having one or more alkyl substituents. Examples of alkaryl groups include, without limitation, tolyl, xylyl, mesityl, ethylphenyl, and methylnaphthyl.
An xe2x80x9carylenexe2x80x9d group is a C6-10 aryl group positioned between and connecting two other substituents. The arylene group may be optionally substituted. A non-limiting example of an arylene group is phenylene. For purposes of the invention, the arylene group preferably constitutes one ring of a fused bicyclic or tricyclic ring system.
A xe2x80x9cheterocyclicxe2x80x9d group or radical is a ring structure having from about 3 to about 8 atoms, wherein one or more atoms are selected from the group consisting of N, O, and S. The heterocyclic group may be optionally substituted on carbon with oxo or with one of the substituents listed above. The heterocyclic group may also independently be substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, or on sulfur with oxo or lower alkyl. Preferred heterocyclic groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholino.
In certain preferred embodiments, the heterocyclic group is a heteroaryl group. As used herein, the term xe2x80x9cheteroarylxe2x80x9d refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 xcfx80 electrons shared in a cyclic array; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of N, O, and S. Preferred heteroaryl groups include, without limitation, thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, thiazolyl, and isoxazolyl. In certain other preferred embodiments, a heterocyclic group is fused to an aryl or heteroaryl group. Examples of such fused heterocyles include, without limitation, tetrahydroquinoline and dihydrobenzofuran.
A xe2x80x9cheteroarylenexe2x80x9d group is a heteroaryl group positioned between and connecting two other groups. The heteroarylene group may be optionally substituted. For purposes of the invention, the heteroarylene group preferably forms one ring of a fused bicyclic or tricyclic ring system.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein refers to chlorine, bromine, fluorine, or iodine.
As herein employed, the term xe2x80x9cacylxe2x80x9d refers to an alkylcarbonyl or arylcarbonyl substituent, wherein the alkyl or aryl portion may be optionally substituted.
The term xe2x80x9camidoxe2x80x9d as employed herein refers to a formylamino, alkylcarbonylamino, or arylcarbonylamino group. The term xe2x80x9caminoxe2x80x9d is meant to include NH2, alkylamino, arylamino, and cyclic amino groups.
The term xe2x80x9cphosphatexe2x80x9d refers to groups in which there are four oxygen atoms around a phosphorous atom. The term xe2x80x9cphosphonatexe2x80x9d refers to groups in which there are three oxygen atoms around a phosphorous atom. The term xe2x80x9cphosph(on)atexe2x80x9d, as used herein refers generally to either a phosphate or a phosphonate. The term xe2x80x9cphosphonylxe2x80x9d refers to a radical in which there are three oxygen atoms around a phosphorous atom. The phosphonyl radical may be attached to carbon to form a phosphonate group or to oxygen to form a phosphate group.
In a first aspect, the invention provides novel acyl phosphate and acyl phosphonate xcex2-lactamase inhibitors. Preferably, such inhibitors have the general mixed anhydride structure (I): 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided, however, that when Y is Z, then Z is not phosphonyl; and further provided that when Y is OZ and Z is phenyl, then X is not methyl or phenyl; when Y is OZ and Z is alkyl or adenosyl, then X is not xcex1-aminoalkyl; and when Y is OZ and Z is benzoyl, then X is not phenyl.
In certain preferred embodiments, the mixed anhydride is an acyl phosphate, and Y is thus OZ. In certain other preferred embodiments, the mixed anhydride is an acyl phosphonate, and Y is thus Z. For purposes of the present invention, the term xe2x80x9cacyl phosph(on)atexe2x80x9d will be used to refer generally to either acyl phosphates or acyl phoshonates.
Z is preferably selected from the group consisting of alkyl, preferably C1-6 alkyl; aryl, preferably C6-14 aryl, more preferably C6-10 aryl, most preferably phenyl or substituted phenyl; aralkyl, preferably (C6-10)ar(C1-6)alkyl, more preferably (C6-10)ar(C1-3)alkyl, most preferably benzyl or naphthylmethyl; acyl, preferably acetyl, benzoyl, or substituted benzoyl; heterocyclic radical, preferably heteroaryl or fused heteroaryl; and phosphonyl.
X is selected from the group consisting of alkyl, preferably C1-6 alkyl; aryl, preferably C6-14 aryl, more preferably C6-10 aryl, most preferably phenyl or substituted phenyl; aralkyl, preferably (C6-10)ar(C1-6)alkyl, more preferably (C6-10)ar(C1-3)alkyl, most preferably benzyl or naphthylmethyl; and heterocyclic radical, preferably heteroaryl or fused heteroaryl. In some preferred embodiments, X is aminoalkyl. In these embodiments, X together with the adjacent xe2x80x94C(O)xe2x80x94Oxe2x80x94 preferably forms an xcex1-amino acid, more preferably a naturally occurring xcex1-amino acid.
Any of the alkyl, aryl, or heteroaryl groups in X or Z may be optionally substituted with one to three, preferably one or two, substituents selected from the group consisting of halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, amino, alkylcarboxamido, arylcarboxamido, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, cyano, and alkylaminocarbonyl. More preferably, an aryl or heteroaryl group in X or Z is optionally substituted with one or two substituents independently selected from the group consisting of C1-4 alkyl, C1-4 alkoxy, C6-10 aryl, and nitro.
In certain preferred embodiments, Y and X are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene, and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted; provided that X is not phenylethene. In certain preferred embodiments, X is a fused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. In one such preferred embodiment, X is phenylene, giving the structure of Formula III: 
wherein Y is O, or alkylene.
In neutral aqueous solution, most compounds of the invention are surprisingly stable, with half-lives for spontaneous hydrolysis of between 0.6 and 80 days. Compounds with electron-withdrawing acyl substituents, such as compound XXIII, or with aliphatic acyl groups, such as compound X, exhibit shorter half-lives. This pattern of reactivity suggests that attack by water/hydroxide occurs at the acyl rather than the phosphyl center. Compounds XXXII and XXIII are more rapidly hydrolyzed.
The acyl phosph(on)ates of the invention are both substrates and irreversible inhibitors of xcex2-lactamases. While not wishing to be bound by theory, the inventors have found that turnover, as a substrate, involves fast acylation followed by slower deacylation, whereas irreversible inactivation involves phosphylation. In general, acylation is far more rapid than phosphylation, and is enhanced by hydrophobic groups in both the acyl and leaving group moieties of the acyl phosph(on)ate. Acylation results in the formation of a tight acyl-enzyme complex, giving rise to reversible enzyme inhibition.
The compounds according to the invention are useful as xcex2-lactamase inhibitors. In certain preferred embodiments, the compounds of the invention also have utility as antibiotic agents. Furthermore, the inhibitors of the invention are also useful as probes for elucidating and identifying mechanisms responsible for bacterial antibiotic resistance and for evaluating the effect of administering agents to overcome such resistance concomitantly with antibiotic treatments.
Nonlimiting examples of certain preferred embodiments according to the invention appear in Table 1. These examples are shown as salts. It would be evident to one skilled in the art that the compounds of Formulae I-III can exist in conjugate acid, conjugate base, or salt form. The disclosure of compounds, compositions, and methods contained herein is, in each instance, expressly intended to include all such forms.
In a second aspect, the invention provides pharmaceutical compositions comprising an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor and a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, such inhibitors have the general mixed anhydride structure (I): 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
Preferred values for X, Y, and Z according to this aspect of the invention are as described above for the first aspect.
In certain preferred embodiments, Y and X are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted. In certain preferred embodiments, X is a fused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. In one such preferred embodiment, X is phenylene, giving the structure of Formula III: 
wherein Y is O or alkylene.
The characteristics of the carrier, excipient, or diluent will depend on the route of administration. As used herein, the term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The term xe2x80x9cphysiologically acceptablexe2x80x9d refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Preferably, the biological system is a living organism, more preferably a mammal, most preferably a human. Thus compositions and methods according to the invention, may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
In certain preferred embodiments, the pharmaceutical compositions according to this aspect of the invention additionally comprise an antibiotic agent. In particularly preferred embodiments, the antibiotic agent is a xcex2-lactam antibiotic. The pharmaceutical composition of the invention may also contain other active factors and/or agents which enhance inhibition of xcex2-lactamase and/or DD-peptidases.
The term xe2x80x9cantibioticxe2x80x9d is used herein to describe a composition which decreases the viability or which inhibits the growth or reproduction of microorganisms. As used in this disclosure, an antibiotic is further intended to include an antimicrobial, bacteriostatic, or bactericidal agent. Non-limiting examples of antibiotics useful according to this aspect of the invention include penicillins, cephalosporins, aminoglycosides, sulfonamides, macrolides, tetracyclins, lincosides, quinolones, chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid, spectinomycin, trimethoprim, sulfamethoxazole, and others. The term xe2x80x9cxcex2-lactam antibioticxe2x80x9d is used to designate compounds with antibiotic properties containing a xcex2-lactam functionality. Non-limiting examples of xcex2-lactam antibiotics useful according to this aspect of the invention include penicillins, cephalosporins, penems, carbapenems, and monobactams.
In certain preferred embodiments, the second aspect of the invention provides pharmaceutical compositions for use in the methods of the invention.
In a third aspect, the invention provides methods for inhibiting in vitro or in vivo xcex2-lactamase activity, such methods comprising administering an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor. Preferably, such inhibitors have the general mixed anhydride structure of Formula I: 
or salts thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
Preferred values for X, Y, and Z according to this aspect of the invention are as described above for the first aspect.
In certain preferred embodiments, X and Y are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted. In certain preferred embodiments, X is a fused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. In one such preferred embodiment, X is phenylene, giving the structure of Formula III: 
wherein Y is O or alkylene.
Non-limiting examples of particularly preferred inhibitors to be used according to this aspect of the invention are shown in Table 2.
In a fourth aspect, the invention provides a method for inhibiting bacterial growth, the method comprising administering an acyl phosphate or acyl phosphonate xcex2-lactamase inhibitor. Preferably, such inhibitors have the general mixed anhydride structure of Formula I: 
or salt thereof;
wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z or OZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, or phosphonyl; provided that when Y is Z, then Z is not phosphonyl.
Preferred values for X, Y, and Z according to this aspect of the invention are as described above for the first aspect.
In certain preferred embodiments, X and Y are taken together with the remaining atoms of the chain to form a cyclic structure of Formula II: 
wherein Y is O or alkylene and X is alkylene, cycloalkylene, fused heterocycle, heteroarylene, or arylene, and wherein the alkylene, cycloalkylene, fused heterocycle, heteroarylene, and arylene groups may be optionally substituted. In certain preferred embodiments, X is a fused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. In one such preferred embodiment, X is phenylene, giving the structure of Formula III: 
wherein Y is O or alkylene.
The methods according to this aspect of the invention are useful for inhibiting bacterial growth in a variety of contexts. For example, such methods can be used to prevent the growth of xcex2-lactam resistant bacteria in experimental cell cultures. Such methods can also be used to prevent the growth of xcex2-lactam resistant bacteria in veterinary contexts. In addition, such methods can be used to prevent the growth of xcex2-lactam resistant bacteria in human patients.
Accordingly, in a preferred embodiment of this aspect, the invention provides methods for inhibiting bacterial growth in an animal, including a human, comprising the step of administering a therapeutically effective amount of xcex2-lactamase inhibitors according to the invention for a therapeutically effective period of time to the animal.
The terms xe2x80x9ctherapeutically effective amountxe2x80x9d and xe2x80x9ctherapeutically effective period of timexe2x80x9d are used to denote known treatments at dosages and for periods of time effective to show a meaningful patient benefit, i.e., healing of conditions associated with bacterial infection, and/or bacterial drug resistance. The xcex2-lactamase inhibitors of the invention may be administered to the animal by any route, including parenterally, orally, sublingually, transdermally, topically, intranasally, intratracheally, or intrarectally. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of inhibitor from about 0.1 xcexcg/mL to about 1 mg/mL, more preferably from about 0.1 xcexcg/mL to about 100 xcexcg/mL, and most preferably from about 0.1 xcexcg/mL to about 10 xcexcg/mL. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. In a preferred embodiment, the inhibitor is administered orally.
In certain preferred embodiments of the method according to this aspect of the invention, a xcex2-lactamase inhibitor according to the invention is co-administered with an antibiotic. In a particularly preferred embodiment of the invention, the co-administered antibiotic is a xcex2-lactam antibiotic. For purposes of this invention, the term xe2x80x9cco-administeredxe2x80x9d is used to denote sequential or simultaneous administration. In certain other preferred embodiments, the xcex2-lactamase inhibitor according to the invention has antibiotic activity, and thus either can be administered alone or can be co-administered with a xcex2-lactam antibiotic or any other type of antibiotic. In some embodiments, more than one xcex2-lactamase inhibitor may be administered sequentially or simultaneously.
The following examples are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.